Abstract C. difficile infection (CDI) is the most common infection patients develop while admitted to hospitals, resulting in an annual three billion dollar cost to the US healthcare system. This bacterial infection of the colon is created by the use of antibiotics which kill commensal bacteria, creating an environment in the large intestine which is conducive to the development of CDI. The conventional treatment for most cases of CDI remains metronidazole or vancomycin, two antibiotics which like all other antibiotics also have indiscriminate effects on the bacteria of the gut. These antibiotics are associated with recurrence rates of CDI ranging from 15-25% following the initial episode of CDI. While the incidence of CDI has increased, and as recurrence rates for CDI have continued to rise, the incidence of life threatening forms of CDI now occur in up to 10% of patients with this infection. While this disease has evolved, the conventional therapies available for CDI have remained virtually unchanged for the previous 20 years. The key to improving CDI treatment is to avoid the disturbances to the bacterial component of the microbiome imposed by conventional antibiotics. Our group has published the first and only paper describing the successful in vitro application of bacteriostatic antisense antibiotics targeting C. difficile. Our previous work utilized dequalinium analogues as cationic bola-amphiphiles (CABs) which are used to complex with and deliver antisense phosphorothioate gapmers into C. difficile to inhibit translation of mRNAs important for bacterial survival. The objective of this study is to develop a new class of antibiotics which are antisense in their action, targeting C. difficile while limiting effects on other bacteria. This current study proposal will accomplish this goal through two aims which use methods well established from the scientific community and quite familiar to our group. In our first aim, our multidisciplinary team will synthesize new CABs which have never been described in the literature and which are rationally designed based upon our team of experts who have a rich experience with nanocarriers. We will develop CABs which are excellent at complexing with our antisense gapmers, which are not toxic to colonocytes and which have minimal to no effect on non-difficile bacteria. In our second aim, we will then develop CAB-gapmer nanocomplexes against C. difficile, with the goal of creating antisense antibiotics which are bacteriostatic against C. difficile in vitro, which have little to no effect on other gut bacteria as tested in vitro, and which have no toxicity to colonocytes. Our overall goal is to create a group of well-characterized antisense nanocomplexes which have been thoroughly tested in vitro, allowing for their subsequent testing in CDI animal models following the conclusion of this project.